Hollow fiber bioreator technology is an economical alternative to traditional cell culture methods for the production of cells and cell-derived products (Hirschel and Gruenberg, "An Automated Hollow Fiber System for the Large Scale Manufacture of Mammalian Cell Secreted Product". In Large Scale Cell Culture Technology; Lydersen, B., Ed.; 1988; pp. 113-144), and is finding increased use for novel applications in the fields of tissue engineering (Maki et al., "Treatment of Diabetes by Xenogenic Islets Without Immunosuppression: Use of a Vascularized Bioartificial Pancreas". Diabetes 1996, 45, 342-347; Sielaff et al., "Gel-Entrapment Bioartificial Liver Therapy in Galactosamine Hepatitis". J Surg. Res. 1995, 59, 179-184), toxicology (Stanness et al., "A Dynamic Model of the Blood-Brain Barrier In Vitro". Neurotoxicology 1996, 17, 481-496), and pharmacology (Lister et al., "Importance of .beta.-lactamase Inhibitor Pharmacokinetics in the Pharmacodynamics of Inhibitor-Drug Combinations: Studies with Piperacillin-Tazobactum and Piperacillin-Sulbactam" in Animicrob. Agents Cheinother, 1997, 41, 721-727; Moore et al., "Activity of (s)-1-(3-Hydroxy-2-Phosphonylmethoxypropyl) Cytosine against Human Cytomegalovirus when Administered as a Single Bolus Dose and Continuous Infusion in In Vitro Cell Culture Perfusion System". Antimicrob. Agents Chemother. 1994, 38, 2404-2408).
A diagram of a simple hollow fiber system is shown in FIG. 1. Cells are typically inoculated into the area outside of the hollow fibers, i.e., into the extracapillary (EC) space. Medium is circulated from a reservoir, through a pump, gas exchange cartridge, in order to pass through the fibers and within the intracapillary (IC) space, to be finally returned to the reservoir. For instance, Yoshida et al. (U.S. Pat. No. 4,391,912) describes a method of cultivating cells that involves the use of culture medium in a bioreactor having a shell and a plurality of hollow fibers, wherein the medium passes through the interior of the fibers and cells are cultivated in the space between the shell and the fibers.
Yet another approach is taken in the line of Tricentric.TM. bioreactors available from Setec, Livermore Calif. These bioreactors employ a concentric "fiber-within-fiber" geometry in which all cells are said to be grown within 100 microns of a nutrient source. More recent advances in this area are described, for instance, in U.S. Pat. No. 5622857, R. Goffe, which describes a high performance hollow fiber bioreactor having concentric hollow fiber bundles, a central hollow fiber bundle supplies media, and an outer array supplies oxygen needed for cell culture. The bioreactor is said to be useful for expanding therapeutic cells such as stem cells ex vivo, and as an extracorporeal device such as an artificial liver.
Typically, the hollow fiber membranes that serve to separate the cells and the circulating medium provide a predetermined molecular weight cutoff, e.g., allowing the passage of molecules having a molecular weight of under about 10 kD, while preventing the passage of larger molecules and cells themselves. Therefore, with cells being maintained in the EC space, nutrients such as glucose and oxygen are delivered in medium and passed through the fibers. The nutrients are able to pass through the fibers to be fed to the cells, while waste products such as lactate and carbon dioxide can pass through as well to be removed through the fibers and diluted in the medium reservoir. Large molecular weight growth factors in serum (or serum-free medium components such as transferring and albumin) can be supplied by the medium directly to the EC space containing the cells. The IC medium reservoir is changed every day or two to provide fresh basal nutrients. The EC medium is also changed every few days to replenish the high molecular weight growth factors and to harvest whatever product may be retained on the EC side along with the cells.
Occasionally, bioreactors have been described in which cells are grown in the IC, rather than EC, space. See, for instance, Sielaff et al. cited above. While these approaches generally provide improved flow distribution, they also suffer from several potential drawbacks as well, including poorer cell retention and reduced cell culture volume.
There are a number of advantages to using hollow fiber perfusion bioreactors for the production of proteins using mammalian cells. These advantages include the direct result of cell retention and the high density cell growth that hollow fiber systems provide. The use of a semi-permeable membrane to retain high molecular weight proteins on the cell side allows for more efficient use of expensive medium components while producing a highly concentrated product. As a result, cost reductions are obtained through continuous production, lower overhead, lower labor, reduced medium costs, and lower purification costs.
Since the presence of an ample supply of oxygen is generally considered the limiting factor in hollow fiber systems (see, e.g., Piret and Cooney, "Model of Oxygen Transport Limitations in Hollow Fiber LBioreactors". Biotech. Bioeng. 1991, 37, 80-92), it is common to provide the bioreactor with both continual IC medium (or EC medium, in the case of IC cell growth) recirculation as well as an in-line gas exchanger. The oxygen supply system is housed in a CO.sub.2 incubator for pH buffering and temperature control. More sophisticated, dedicated systems include additional process controls such as automatic pH control, continuous medium replacement, and other strategies to optimize production (Hirschel and Gruenberg, cited above).
A continuing weakness of hollow fiber technology is an inherent lack of predictability, since there is no efficient screening method to determine how well a new cell line will perform in a hollow fiber system, or how well an established cell line will perform under new conditions. Instead, it is usually necessary to go through the costly and time consuming processing of complete scale up, on a cell line by cell line basis, in order to determine performance. Commercially available bioreactors are too large and expensive for simple screening protocols. Even small, inexpensive bioreactors are not well-suited for screening protocols since a pump is required to support the oxygen demand for each bioreactor.
Data from 96-well plates or T-flask cultures are useful for predicting cell line performance in stirred-tank reactors. Such data, however, does not correlate well with cell growth and productivity in high density hollow fiber cultures (Schlapfer et al., "Development of Optimized Transfectoma Cell Lines for Production of Chimeric Antibodies in Hollow Fiber Cell Culture Systems", Biotech. Bioeng., 1995, 45, 310-319). Research to understand the fundamental mechanisms that affect cell productivity in hollow fiber culture is very complex, especially since several potentially critical factors are likely to be cell line dependent. How a particular cell line will react, for instance, to the partition of medium components across the membrane and to other stresses of high density culture is not well-understood.
This lack of understanding is due, in part, to the lack of a good model system for fundamental research and process development. One approach to hollow fiber research is to use a small system such as that shown in FIG. 1 (Jackson et al., "Evaluation of Hollow Fiber Bioreactors as an Alternative to Murine Ascites Production for Small Scale Monoclonal Antibody Production", J. Immunol. Methods 1996, 189, 217-231; Schlapfer et al., cited above). However, these systems are still too expensive for routine development, and cell growth takes several weeks to reach confluency. As a result, research proceeds very slowly, and replicates are often omitted so that the significance of the results is unknown. Very small hollow fiber bioreactors with only one or a few fibers have been used for fundamental research (Sardonini and Dibiasio, "Investigation of the Diffusion-Limited Growth of Animal Cells Around Single Fibers". Biotech Bioeng. 1992, 40, 1233-1242). However, each bioreactor still requires an independent flow circuit and medium recirculation pump to meet the cellular oxygen demand. As a result, this type of system is not well-suited for use as a screening tool.
What is clearly needed is a method and apparatus for screening cell lines in a manner that will inexpensively and quickly predict their use in a hollow fiber system.